WO2018096212A1

WO2018096212A1 - Arctic bramble (rubus arcticus) cell cultures, method for producing arctic bramble cell cultures, compositions comprising arctic bramble cell cultures and use of arctic bramble cell cultures

Info

Publication number: WO2018096212A1
Application number: PCT/FI2017/050808
Authority: WO
Inventors: Liisa Nohynek; Riitta PUUPPONEN-PIMIÄ; Kirsi-Marja Oksman-Caldentey; Heiko Rischer
Original assignee: Teknologian Tutkimuskeskus Vtt Oy
Priority date: 2016-11-24
Filing date: 2017-11-22
Publication date: 2018-05-31
Also published as: KR20190089012A; EP3544411A1; FI20165897A; KR102646959B1; EP3544411A4; FI20165897L; FI128318B

Abstract

The present invention relates to a method for producing artic bramble cell culture, said method comprising two stages, where in the first stage arctic bramble callus line is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, under day-night irradiation regime under normal light or under irradiation using LED light carried out as day-night irradiation or as constant irradiation, to obtain primary arctic bramble suspension cell culture, and in the second stage the primary arctic bramble suspension cell culture is up-scaled in at least two steps in the liquid medium; and irradiation with LED light is used in the first stage or in the second stage or in both stages.

Description

ARCTIC BRAMBLE (RUBUS ARCTICUS) CELL CULTURES, METHOD FOR PRODUCING ARCTIC BRAMBLE CELL CULTURES, COMPOSITIONS COMPRISING ARCTIC BRAMBLE CELL CULTURES AND USE OF ARCTIC BRAMBLE CELL CULTURES

FIELD OF THE INVENTION

The present invention relates to a method for producing artic bramble cell cultures. The invention relates further to arctic bramble cell cultures obtained by the method. The invention relates also to compositions comprising arctic bramble cell cultures, and to the use of the arctic bramble cell cultures in cosmetic, hygiene and personal care applications, in food, nutrition, nutraceuticals, pharmaceutical products, and for providing aroma, flavor and/or color to the products.

BACKGROUND OF THE INVENTION

Arctic bramble (Rubus arcticus) is a unique arctic berry with special chemical and flavor components, which are exploited by food and beverage industry. Arctic bramble is a rare aromatic berry species. It is rich in phenolic compounds, such as tannins and anthocyanins with known beneficial effects to the human health. However, the availability and quality of wild berry material is strongly affected by seasonal variations and by chemical and biological pollutants. The annual crop of both wild and cultivated berries is extremely low, particularly because these berries are very sensitive to climate changes and plant pathogenic microbes, and they are difficult to cultivate. Therefore, the availability of berry material for industry is insufficient.

The cosmetic industry uses various plant-derived fractions due to their bioactivities in cosmetic products. Many cosmetic companies are constantly looking for more advanced products for the markets, such as products containing plant cell cultures, for providing bioactive effects. Plant metabolites are typically very complex, and therefore their chemical synthesis may be difficult or even impossible. Plant cells and tissue cultures have been recognized as potential options for replacing whole plants as sources of valuable industrial plant bio-chemicals. Undifferentiated plant cells are totipotent, containing the full complement of genetic information, and therefore have the capacity to develop into any organ of the plant. Plant callus is a mass of somatic cells that undergo dedifferentiation to give rise to totipotent embryogenic cells, which temporarily gain the ability to proliferate and/or regenerate an embryo. Callus and plant stem cells are fundamentally different from each other, despite the fact, that callus exhibits some stem cell-like properties for a temporary period, and that it has been cultured for useful plant compounds as an alternative source of plant stem cell.

Callus is similar as plant stem cell in its ability to differentiate, but the two are different in their origin. While plant stem cell exists in the meristematic tissues of plant, callus is obtained as a temporary response to cure wound in somatic cell. Only plant stem cells embedded in meristems can divide and give rise to cells that differentiate while giving rise to new stem cells. Cultured callus-derived cells provide a cost-effective and sustainable source of important natural products.

Cloudberry cell cultures have been studied and methods for their production have been proposed. WO 2013/124540 Al discloses cosmetic compositions containing cloudberry cell culture preparation, where said compositions have antioxidant and anti-aging effect. It protects the skin from UV radiation effects and it has effect on the procollagen I synthesis of aged fibroblasts. In said method callus is first produced from sterile cuts of cloudberry plant. The material is maintained on a medium favoring continuous growth of the non- differentiated cells. Selected callus is then chosen for suspension culture, which is stepwise scaled up to large scale cultivation in a bioreactor. The biomass is harvested, followed by washing with water and freeze-drying. The freeze-dried powder may be used as such in cosmetic compositions or the powder may be extracted with methanol or ethanol and filtered prior to use.

Despite the ongoing research and development in the field, there is a need for new sustainable and ecological approaches to provide valuable and unique cell cultures originating from arctic bramble.

SUMMARY OF THE INVENTION

Cell culture technology offers a sustainable approach for utilizing the biosynthetic capacity of arctic bramble plant to produce valuable phenolic metabolites. Even new compounds, which cannot be found in the natural plant and berries, can be produced utilizing modified biosynthetic pathways.

An object of the invention is to provide a method for producing arctic bramble cell cultures. Another object of the invention is to provide a method for producing arctic bramble cell cultures, said method being suitable particularly for larger industrial scale. Another object of the invention is to provide compositions comprising said arctic bramble cell cultures.

An object of the invention is to provide arctic bramble cell cultures. Another object of the invention is to use of the obtained arctic bramble cell cultures in cosmetic, hygiene and personal care products, as well as for food, nutrition, nutraceuticals, pharmaceuticals and as food flavors and colorants.

The present invention generally concerns producing arctic bramble cell cultures having unique compositions, where cell cultures are illuminated with LED-lights favoring strong production of anthocyanins in the culture.

The method of the invention for producing arctic bramble cell culture comprises two stages, where in the first stage arctic bramble callus line is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, under day-night irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation, to obtain primary arctic bramble suspension cell culture, and in the second stage the primary arctic bramble suspension cell culture is up-scaled in at least two steps in the liquid medium; and irradiation with LED lights is used in the first stage or in the second stage or in both stages.

The invention also relates to compositions comprising arctic bramble cell cultures obtained by the method, said arctic bramble cell cultures comprising one or more of anthocyanins, procyanidins, phenolic acids, vitamins and fatty acids, beneficial for human health.

The invention further relates to the use of said arctic bramble cell cultures in cosmetic compositions, hygiene and personal care products, as well as in foods, feeds, pet foods nutritional compositions, nutraceutical compositions, pharmaceutical compositions, for example for providing effects beneficial to the health, and as providing flavor, aroma and/or color to the products.

Characteristic features of the invention are presented in the appended claims. DEFINITIONS

The term "cell culture" refers here to callus or cell suspension culture or hairy root originating from arctic bramble (Rubus arcticus) plant, where said plant includes berries, seeds, flowers, roots, leaves and pieces of plant stem, one or more of hypocotyl, cotyledon, leaf section, stem section, root section of a seedling; stem section including a node or an internode, and a leaf section of a mature plant.

The term "MS medium" refers here to Murashige and Skoog medium having a pH of 5.8, widely used in the field of plant cell and tissue culture. The MS medium may be modified, where the MS medium is supplemented with additions of sucrose, plant growth regulators, also called plant growth hormones, the pH may be adjusted, and for obtaining solid medium agar or gelrite may be added.

LED light carried out as intermitting irradiation refers here to illumination/irradiation carried out in intermittent periods with LED light irradiation (light period) and without LED light irradiation (dark period), where the dark period may be from 1 to 23 hours and the light period may be from 1 to 23 hours. An example of intermitting irradiation comprises irradiation carried as day-night irradiation (16/8hours).

Normal light refers here to incandescent light having illumination/irradiation intensity of 50-300 Ix.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows anthocyanin contents of arctic bramble cell cultures obtained with the method of the invention using the specified irradiation with LED lights and without it.

DETAILED DESCRIPTION OF THE INVENTION

Arctic bramble cell culture may be established from wild or cultured arctic bramble plant materials. Examples of such materials are wild arctic bramble plants. Said plants may be collected for example from the northern region in Finland.

Callus may be produced from sterile pieces of in vitro grown arctic bramble plants, generated from sterilized pieces of the plant, or alternatively from surface sterilized pieces of arctic bramble plant materials. Preferably sterile pieces of in vitro grown arctic bramble plants, generated from sterilized pieces of the plant are used in the method. The callus is grown in vitro at sterile conditions, and it is maintained on a medium favoring continuous growth of the non-differentiated cells. Different callus lines (cell cultures) may be separated based on callus color. Particularly suitably bright colored callus of uniform quality is selected by sub-culturing. Typically, at least six different intensely colored callus lines may be selected, stabilized and maintained.

At least one selected callus line is suitably chosen for suspension culture. In the present invention, at least one selected arctic bramble callus line (such as intensely colored callus line) is suspension cultured. The suspension culture is up-scaled to produce the arctic bramble cell cultures in amounts suitable for large industrial scale.

The obtained cell culture is suitably cryo-preserved to provide a stable powdery product.

It was surprisingly found that using the suspension culture method of the invention arctic bramble cell cultures having several advantageous effects could be obtained effectively and consistently on a large industrial scale. The obtained arctic bramble cell cultures exhibit valuable bioactivities and other properties. The method is sustainable and continuously large amounts of the arctic bramble cell culture may be produced.

The phenolic profiles of the obtained arctic bramble cell cultures are unique and different compared to the arctic bramble berry fruit. High amounts of natural colorants, i.e. anthocyanins and other valuable phenolic compounds may be produced with the method, in significant amounts. The cell cultures also contain natural aromas, flavors and lipids with beneficial fatty acid composition, which are desired ingredients for example in cosmetic, nutraceutical, nutritional and hygiene preparations. The cell cultures can be generated around the year, on an industrial scale, with consistent quality and composition, which can be constantly monitored.

Arctic bramble plant material

The arctic bramble (Rubus arcticus) plant material refers here to berries, seeds, flowers, roots, leaves and pieces of plant stem of arctic bramble plant, including one or more of hypocotyl, cotyledon, leaf section, stem section, root section of a seedling; stem section including a node or an internode, and a leaf section of a mature plant. Said plant material may originate from wild plants or cultivated plants. Formation of calli

Callus may be produced from sterile pieces of in vitro grown arctic bramble plants, generated from sterilized pieces of arctic bramble plant material, or alternatively from surface sterilized pieces of arctic bramble plant material. Preferably callus produced from sterile pieces of in vitro grown arctic bramble plants, generated from sterilized pieces of arctic bramble plant material is used. Calli formed on arctic bramble explants (cell callus of arctic bramble cells derived from arctic bramble plant) is obtained.

In the producing of callus the plant material is suitably cut into pieces. The pieces may have size of about 0.5 - 10 cm.

The pieces of the plant material are treated with at least one surface sterilizing solution, preferably with an ethanol solution followed by hypochlorite solution, to obtain surface sterilized pieces. The ethanol solution comprises suitably at least 50 % v/v, preferably at least 65 % v/v of ethanol in water. The hypochlorite solution comprises from 1 to 3 % by weight of hypochlorite in water. Suitably the surface sterilized pieces are rinsed with sterile water and dried.

The surface sterilized (sterile) pieces may be used for the formation of the calli, however preferably sterile pieces of in vitro grown arctic bramble plants are used for the formation of the calli.

In an embodiment, the surface sterilized pieces are transferred to a solid medium used for plant cell and tissue culture, without plant growth regulators, followed by incubating the pieces with the medium under sterile conditions, at the temperature of 20 - 28°C, under day-night illumination period under normal light (light-dark irradiation regime), for the time of 1 - 4 weeks, whereby roots and new leaves (in vitro grown arctic bramble plants) formed on the arctic bramble explants are obtained. Typically, the day-night photoperiod under normal light refers to 16 : 8 photoperiod with irradiation 30-60 μιηοΙ/ιη²5. Preferably the solid medium used for plant cell and tissue culture is MS medium, with added sucrose and agar. Typically, 3% w/vol of sucrose is added and the medium is solidified with agar. Suitably the incubation is carried out in sealed sterile boxes. Preferably the temperature is in the range of 20 - 25°C. Suitably the incubation is carried out for 1 -3 weeks.

Sterile pieces of the in vitro grown arctic bramble plants, generated from sterilized pieces of the plant material, or pieces of surface sterilized plant material are incubated on a solid medium, suitably MS medium, without or alternatively with plant growth regulators (PGR) favoring development of undifferentiated plant material called callus.

The callus is grown in vitro at sterile conditions, and it is maintained on a medium favoring continuous growth of the non-differentiated cells, suitably MS medium. Different callus lines (cell cultures) may be separated based on callus color.

In an embodiment the sterile pieces of the in vitro grown arctic bramble plants, or the surface sterilized pieces of arctic bramble plant, are transferred to a solid medium used for plant cell and tissue culture, with plant growth regulators, followed by incubating the pieces with the medium, under sterile conditions, at the temperature of 20 - 28°C, under day- night (light-dark) irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation, for the time of 1 - 8 weeks, at the pH in the range of 3 - 7, whereby callus formed on arctic bramble explants is obtained. Preferably LED light is used, particularly preferably intermitting irradiation with LED light.

The day-night (light-dark) irradiation regime with normal light refers typically to a photoperiod 16 : 8 with irradiation 30-60 μιηοΙ/ιη²5. The LED light or lights as defined on page 11 of this specification are preferably used.

Preferably the temperature is 20 - 25°C.

Preferably the incubation time is 1 - 4 weeks.

Preferably the pH is in the range of 4 - 6, particularly preferably 4 - 5.

Preferably the medium is a modified MS medium, where sucrose, agar or gelrite and plant growth regulators are added and where the pH is adjusted into the range of 3-7, preferably of 4 - 6. Suitably said modified MS medium comprises 3 % w/v sucrose and 8 g^L~l Bacto agar and plant growth regulators.

The plant growth regulators (PGRs, plant growth hormones) are selected from cytokines, auxins and combinations thereof. Suitably the cytokine is kinetin, and a suitable auxin is a-naphthaleneacetic acid (NAA). Suitably 0.46 μΜ kinetin and 5.37 μΜ NAA is used. The calli formed on the arctic bramble explants are separated from the explants and transferred to a fresh solid medium, preferable same as used in the forming of callus, and sub-cultured regularly favoring soft productive biomass. Cultured plant cells are totipotent, and when treated with PGRs the cells multiply continuously producing biomass consisting of identical cells.

Different callus lines are separated based on the color of the callus, for sub-culturing.

The sub-culturing is carried out suitably as follows. The calli formed on the arctic bramble explants are separated from the explant and transferred to fresh solid medium and maintained under day-night irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation, preferably LED light is used, particularly preferably intermitting irradiation with LED light. During each sub-culturing the medium is replaced with fresh medium with pH adjustment to 3-7, preferably to 4-6, particularly preferably to 4-5. The separated callus is sub-cultured every 2-6 weeks, suitably by gentle division, for 2 to 8 months to obtain sub-cultured callus line. Any dark brown material is removed Preferably the solid medium is a modified MS medium, where sucrose, agar or gelrite and plant growth regulator(s) are added and where the pH is adjusted into the range of 3-7, preferably of 4 - 6., Preferably at least one PGR selected from cytokine, auxins and combinations thereof is included in said medium. The temperature of 20 - 28°C, preferably 20-25°C is used. Sub-cultured callus lines are obtained.

The day-night (light-dark) irradiation regime with normal light refers typically to a photoperiod 16 : 8 with irradiation 30-60 μιηοΙ/ιη²5.

The LED light or lights as defined on page 11 of this specification are preferably used. Method for producing arctic bramble cell cultures

The method of the invention for producing arctic bramble cell cultures is particularly suitable for larger industrial scale, providing arctic bramble plant cell culture of consistent quality in high amounts. Up-scaling of the primary arctic bramble suspension cell culture is realized by suspension culturing carried out in at least two steps.

The method of the invention for producing arctic bramble cell cultures comprises two stages, where the first stage comprises generation of primary arctic bramble suspension cell culture, where arctic bramble callus line is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, under day-night irradiation under normal light, or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation, to obtain primary arctic bramble suspension cell culture, and in the second stage the arctic bramble suspension cell culture is up-scaled in at least two steps, and irradiation with LED light is used in the first stage or in the second stage or in both stages.

In a preferable embodiment, the up-scaling comprises at least three steps. In another preferable embodiment, the upscaling comprises at least four steps.

Preferably LED light is used, particularly preferably intermitting irradiation with LED light.

In a preferable embodiment LED light carried out as day-night irradiation is used. Day- night irradiation refers typically to a photoperiod of 16: 8 respectively.

First stage: Generation of primary arctic bramble suspension cell culture

In the first stage of the method generation of primary arctic bramble suspension cell culture is carried out. The first stage of the method comprises generation of primary arctic bramble suspension cell culture, where arctic bramble callus line is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, to generate inoculum for larger scale. For carrying out the suspension culture at least one arctic bramble callus line is introduced in a liquid medium comprising a medium used for plant cell and tissue culture, and water, said liquid medium having a pH of 3-7, at the temperature of 20 - 28°C, under day-night irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation preferably LED light is used, particularly preferably intermitting irradiation with LED light. The suspension culture is initiated suitably with 0.1- 20 g of a selected arctic bramble callus line in 10-200 ml of the liquid medium. The suspension culture is sub-cultured every 7-15 days intervals and stepwise up-scaled to 50-500 ml, preferably to 200-500 ml culture in a suitable vessel, such as an Erienmeyer flask. Suitably more than one replicate suspensions are cultured in the flasks, preferably at least two replicate suspensions. The sub-culturing is carried out in the liquid medium as defined below. Any visually detectable clumps are removed, preferably after 2 - 4 sub- culturing, to provide a homogeneous suspension by visual inspection. After 2-10, preferably after 4-8 sub-culturing, primary arctic bramble suspension cell culture is obtained as a finely divided and homogeneous suspension, when inspected visually. In an embodiment, the cells may be harvested using methods known in the art, and suspended in the fresh medium to obtain a homogeneous suspension. The primary arctic bramble suspension cell culture (inoculum) is used in the second stage. The water used in the method is selected from sterilized MQ-water, RO-water, and tap water, preferably high purity sterilized MQ-water or RO-water.

Any medium used for plant cell and tissue culture may be used in the method, preferably the medium used for plant cell and tissue culture is a modified MS medium. Said modified MS medium comprises sucrose, suitably 3 % w/v. Said modified MS medium comprises plant growth regulators selected from cytokines, auxins and combinations thereof. Suitably the cytokine is kinetin, and a suitable auxin is a-naphthaleneacetic acid (NAA). Suitably 0.46 μΜ kinetin and 5.37 μΜ NAA (a-naphthaleneacetic acid) is used. The pH of modified MS medium is adjusted into the range of 3-7, preferably of 4 - 6.

The day-night (light-dark) irradiation regime under normal light refers typically to a photoperiod 16 : 8 with irradiation 30-60 μιηοΙ/ιη²5.

Preferably the temperature is 22-25°C.

The first stage is suitably carried out in a vessel comprising glass or plastic allowing the irradiation pass through the walls of the vessel. Suitably glass flasks used in the art are selected, such as Erlenmeyer flasks etc., or alternatively plastic flasks or bags may be used, which allow passing of the irradiation to the mixture/suspension.

The LED light or lights as defined on page 11 of this specification are preferably used.

Second stage: Up-scaling of the primary arctic bramble suspension cell culture

The second stage of the method comprises up-scaling of the primary arctic bramble suspension cell culture (inoculum from the first stage), where the up-scaling is carried out in at least two steps.

In the first step, the inoculum obtained from the first stage is sub-cultured, suitably every 10±5 days, and up-scaled to a volume of not more than 1000 ml. Suitably a vessel of 200- 1000 ml is used, preferably of 200-500 ml containing about 30 to 80 ml of the primary arctic bramble suspension culture, using the same medium and temperature as in the first stage. During this first step the culture is exposed to no irradiation, or irradiated using day- night irradiation regime using normal light or using LED light carried out as intermitting irradiation or as constant irradiation, to yield cell culture from the first step. Preferably the cells are harvested using methods known in the art, and suspended in the fresh medium to provide an inoculum .

In the second step the inoculum from the first step is up-scaled to a volume of not more than 20 I and made up to with the fresh medium to obtain initial cell density of 10-50 g L^~ ¹, preferably 25-40 g L ^_1. Suitably a bio-reactor having the volume of 1-20 L, preferably 1- 10 L is used . The suspension is grown in the bio-reactor for 7- 15 days, with optionally from 1 to 3 additions of the fresh medium . Suitably cultivation conditions, where the temperature is 20-28°C, preferably 22-26°C are used . Suitably the dissolved oxygen (DO) is at least 10%, preferably at least 20 %. Agitation speed, depending on cultivation vessel, is in the range of 20 to 300 rpm . The pH is suitably uncontrolled . The aeration of 0.25 - 0.5 I min- 1 may be used . A suspension cell culture from the second step is obtained . Preferably the same medium as in the first stage is used . During this second step the culture is irradiated using day-night irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation, preferably LED light is used . In an embodiment where the second stage comprises more than two steps, the second step may be carried out exposed to no irradiation .

The suspension cell culture from the second step may be obtained as the final product or alternatively it may be used as inoculum in a third step. The cells may also be ha rvested using methods known in the art using methods known in the art and the harvested cells may be suspended in the fresh medium to provide the inoculum.

The second stage may also comprise a third step. In the third step the inoculum from the second step is up-scaled to volume of not more than 70 I, made up to with the fresh medium to obtain initial cell density of 10-50 g L ^_1, preferably 25-40 g L ^_1, suitably using a bio-reactor having the volume of 10-70 L, preferably 10-50 L. The suspension is grown in the bio-reactor for 7- 15 days, with optionally one addition of fresh medium. Suitably cultivation conditions, where the temperature is 20-28°C, preferably from 22 to 26°C, and dissolved oxygen (DO) at least 10%, preferably at least 20 % are used . The agitation speed, depending on cultivation vessel, is suitably in the range of 20 - 150 rpm . The pH is suitably uncontrolled . The aeration is preferably 0.3 - 2 I min ^_1 The pressure is suitably in the range 0.008 to 0.2 bar, depending on cultivation vessel . Preferably the same medium as in the first stage is used . During this third step the culture is irradiated using day-night irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation, preferably LED light is used, particularly preferably intermitting irradiation with LED light. In an embodiment where the second stage comprises more than three steps, the third step may be carried out exposed to no irradiation.

A suspension cell culture from the third step is obtained as the final product or alternatively it may be used as inoculum in a fourth step. The cells may also be harvested using methods known in the art. The harvested cells may be suspended in the fresh medium to provide the inoculum.

The second stage may also comprise a fourth step. In the fourth steps the inoculum from the third step is up-scaled to volume of not more than 500 I made up to with fresh MS medium to obtain initial cell density of 10-50 g L ^_1, preferably 25-40 g L ^_1, suitably using a bio-reactor having the volume of 300-500 L. The suspension is grown in the bio-reactor for 7-15 days, with optionally one addition of fresh medium, which may comprise 2- to 4- fold by weight of sucrose. Suitably cultivation conditions, where the temperature is from 20-28°C, preferably 22 to 26°C, and dissolved oxygen (DO) at least 10 %, preferably at least 20 % are used. The agitation speed is suitably in the range of 50 - 100 rpm. The pH is suitably uncontrolled. The aeration is 10 - 20 I miir¹. The pressure is preferably 0.2 bars. Preferably the same MS medium as in the first stage is used. During this fourth step the culture is irradiated using day-night irradiation regime under normal light or under irradiation using LED light carried out as intermitting irradiation or as constant irradiation preferably LED light is used, particularly preferably intermitting irradiation with LED light. A suspension cell culture comprising biomass is obtained. The cells may also be harvested using methods known in the art. The harvested cells may be suspended in the fresh medium.

The second stage may also comprise a further up-scaling steps. In the method of the invention the final product suspension cell culture, comprising the biomass, obtained in the second stage, may be used as such, or alternatively it may be dried, suitably using cryo-drying or freeze-drying methods known in the art to provide a finely divided powder. Alternatively, the cells may also be harvested using methods known in the art, and used as such or dried. The pH of the MS medium used in the method may preferably be adjusted to 3.5-5, in all steps before autoclaving the medium, using 0.1 M HCI.

The first step is suitably carried out in a vessel comprising glass or plastic allowing the irradiation pass through the walls of the vessel. Suitably glass flasks used in the art are selected.

The second and further up-scaling steps may be carried out in vessels, such as bioreactors allowing the cultivation to final culture volume and passing of the irradiation to the reaction mixture. Suitably vessels comprising glass or plastic material may be used.

LED lig ht

LED (light emitting diode) light is used in the method. Preferably the LED light is used in all stages and all steps. The LED light has color temperature of 2700-3000K.

In an embodiment, the LED light comprises an effectual spectrum comprising at least 25 % of an integral in the wave length range of 630 nm-760 nm (red light integral), calculated from the total effectual spectrum. Red light is generally classified as "warm light shade".

In an embodiment, the LED light comprises a total effectual spectrum in the wave length range of 400-800nm.

In an embodiment, the LED light comprises less than 75 % of an integral in the wave length range of 400-450 nm (violet light integral) or of an integral in the wave length range of 450-490 nm (blue light integral) or of an integral in the wave length range of 490-560 nm (green light integral) or of an integral in the wave length range of 560-590 nm (orange light integral) or a combination thereof. In the method irradiation source is preferably LED light with illumination intensity over 500 Ix, preferable over 800 Ix.

One LED light (source) or combination of LED lights (light sources) may be used .

Said LED lights may be arranged in a panel surrounding or enclosing the reaction vessel, or in any other way to provide the reaction mixture the required irradiation. The final product suspension cell culture, comprising the biomass, obtained in the second stage, may be used as such in the manufacture of products, or alternatively it may be dried, suitably using cryo-drying or freeze-drying methods known in the art to provide a finely divided powder. Alternatively, the cells may also be harvested, and used as such or dried. The biomass may be separated using technique known in the art. Suitably a filter press or vacuum filtration is used, where the biomass is filtered and washed with sterile water. The biomass filter cake is suitably dried, for example using freeze-drying. The dry biomass may be ground to finely divided powder and stored in sealed packages, preferably at temperatures of about -20°C.

Arctic bramble cell cultures are obtained.

Arctic bramble cell cultures

The arctic bramble cell cultures obtained with the method of the present invention exhibit high bioactivities and other beneficial and interesting properties.

The chemical composition of the cell cultures obtained by the method is clearly different from the one natural arctic bramble fruit. Using the irradiation with the specific LED light in the manufacturing method, high amounts of cell lines with very intense red, yellow, orange, green or purple color and unique chemical composition can be obtained in a consistent manner.

Natural arctic bramble berry fruit contains typically hydroxycinnamic acids and their derivatives, anthocyanins, flavonols, ellagic acid derivatives and ellagitannins, gallic acid derivatives and cathechins. Procyanidins are not found in the natural plants. The arctic bramble cell cultures obtained with the method may comprise 0.1-10 wt% (dry weight) of anthocyanins and 0.1-5 wt% (dry weight) of proanthocyanidins. The arctic bramble cell cultures obtained by the method of the invention comprise surprisingly significant amounts of anthocyanins, and flavonoids, such as quercetin derivatives, kaempherol derivatives, prodelphidins and procyanidins having high degree of polymerization, typically at least 13. Further, said arctic bramble cell cultures comprise typically about 200-400 mg/g of proteins, fatty acids such as a-linoleic acid and carbohydrates, such as sucrose. Anthocyanins exhibit antioxidant and anti-inflammatory effect, in addition to intensive color, aroma and flavor, and thus they are beneficial for use in cosmetic, hygiene and personal care applications, in food, feeds, pet foods, nutritional products, nutraceuticals, pharmaceutical products, and for providing aroma, flavor and/or color to the products. Antioxidant activity is also essential for example in delaying skin aging processes. With the method of the invention the anthocyanin content can be increased over seven-fold, particularly with the red-colored arctic bramble cell lines, compared to the manufacture under normal light conditions, and further, the content of other phenolic compounds is increased, especially the content of quercetin and procyanidin. The cells obtained with the method are more intact and contain significantly less water when compared with the ones manufactured under normal light conditions. They provide an advantage as being more effective and economic in downstream processing.

The phenolic compounds, such as flavonoids exhibit antimicrobial activity. Antimicrobial activity is beneficial in cosmetic applications, where for example in cosmetic products it helps to balance skin microbiota and prevents product contaminations.

The invention provides compositions comprising arctic bramble cell cultures obtained by the method, said arctic bramble cell cultures comprising anthocyanins, procyanidins, phenolic acids, vitamins and fatty acids beneficial for human health.

The invention relates also to compositions comprising arctic bramble cell cultures obtained by the method of the invention, where the composition is selected from cosmetic compositions, hygiene and personal care products, as well as in foods, feeds, pet foods nutritional compositions, nutraceutical compositions, and pharmaceutical compositions.

The invention relates also to the use of said arctic bramble cell cultures in compositions selected from cosmetic compositions, hygiene and personal care products, as well as in foods, feeds, pet foods nutritional compositions, nutraceutical compositions, and pharmaceutical compositions.

The cosmetic compositions may contain cosmetically acceptable substances in addition to said arctic bramble cell culture(s). Said compositions may be used as day creams, foundation creams, peeling creams, lipsticks, color cosmetics, skin serums, mascaras, products for hair and/or scalp care, washing products for skin or hair, and as products for skin hygiene. The cosmetic composition can be, for example, an emulsion cream, such as a day cream or a foundation cream; peeling cream; a lipstick; a skin serum or a hair cosmetics product, such as a composition for hair conditioning or a composition for scalp treatment.

The cosmetic composition may contain the arctic bramble cell culture in powder form in an amount of 0.001 to 25 % by weight, more preferably the amount of the powder is 0.01 to 5 % by weight, and most preferably 0.01-1 % by weight.

The emulsion cream containing arctic bramble cell culture can be of the type oil-in-water emulsion, water-in-oil emulsion, water-oil-water emulsion or a micro-emulsion. The emulsion cream composition may contain the arctic bramble cell culture in powder form, preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 1 % by weight.

In the emulsion creams the arctic bramble cell culture can be combined also with synthetic or natural vitamins or their combinations. Examples of these vitamins are retinol and A- vitamin palmitate. On the other hand, also different E-vitamins and E-vitamin derivatives, like tocopherol, C-vitamin and its derivatives, like ascorbyl palmitate and magnesium ascorbyl phosphate, panthenol and other B-vitamins and/or biotin may be used . The composition may contain synthetic and/ or natural vitamins preferably 0.01 to 10 % by weight, more preferably 0.02 to 5 % by weight. The emulsion cream compositions may contain, in addition, one or more adjuvants acceptable in the field of cosmetics, such as preservation agents, thickening agents, moisturizing agents, and other suitable additives, such as for example perfumes and/or coloring agents. Suitable preservation agents are for example parabens, phenoxyethanol, imidazolidinyl urea and methyldibromo glutaronitrile. These preservation agents can be used alone or combined with each other. Any thickening agent suitable in the field of cosmetics can be used provided, that it is compatible with the other components of the composition, for example xanthan gum and hydroxyethylcellulose, hydroxypropylmethylcellulose, Sclerotium gum, Chondrus Crispus, polyacrylates, polyacrylamides, cetearyl dimethicone crosspolymer and magnesium aluminium silicate. Thickening agents can be used alone or in combination with each other. Suitable moisturizing agents are for example heptyl undecylenate, hyaluronic acid or sodium PCA. In the emulsion creams, also different skin conditioning agents, for example tocopheryl acetate and ethylhexylglycerin may be used. In addition, one or more compounds acting as an emulsifier, i.e. a compound dispersing and stabilizing the oil in water may be needed in the emulsion cream composition. Useful emulsifiers are all non-ionic emulsifiers accepted by the cosmetic legislation, such as, for example, glyceryl stearate, PEG-5 glyceryl stearate, PEG-1 00 stearate, PEG-30 dipolyhydroxystearate, lecithin, hydrogenated lecithin and PEG- 8 bees wax, steareth-21, steareth-2, sorbitan olivate as well as a mixture of a fatty glucoside, such as for example cetearyl, cocoyl, or myristyl glucoside and a fatty alcohol, such as for example cetearyl, cetyl, stearyl, octyldodecanol, caprylic/capric triglyceride or myristyl alcohol. In addition, of anionic emulsifiers, for example stearic acid, sodium hydroxide and triethanolamine are useful. Also, different chelating agents, like disodium EDTA and citric acid may be used.

The peeling cream compositions contain arctic bramble cell culture preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 1 % by weight.

The peeling cream compositions may contain in addition to arctic bramble cell culture also other substances, which are acceptable in the field of cosmetics and which are traditional components of peeling cream compositions. Peeling cream compositions can also contain emulsifiers like PEG-8, behenyl alcohol, arachidyl glucoside and arachidyl alcohol, thickening agents like ethylcellulose and various vitamins and derivatives of those, like tocopherol, ascorbyl palmitate and ascorbic acid. The peeling cream compositions can in addition contain one or more adjuvants and/or additives acceptable in the field of cosmetics, such as preservation agent. Suitable preservation agents are for example parabens and phenoxyethanol. These preservation agents can be used alone or in combination with each other. The lipstick compositions contain arctic bramble cell culture preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 2 % by weight.

The lipstick compositions may contain in addition to arctic bramble cell culture also other substances, like different waxes, oils, coloring and pearlescent agents, which are acceptable in the field of cosmetics and which are traditional components of lipstick compositions. Usable waxes are the natural waxes, such as bees wax, candelilla, carnauba, cereal based waxes, jojoba wax and their derivatives, in addition paraffin waxes and synthetic polyethylenes. Lipstick compositions can also contain emulsifiers like PEG-8, behenyl alcohol, arachidyl glucoside and arachidyl alcohol, thickening agents like ethylcellulose and various vitamins and derivatives of those, like tocopherol, ascorbyl palmitate and ascorbic acid. The lipstick compositions can in addition contain one or more adjuvants and/or additives acceptable in the field of cosmetics, such as preservation agent. Suitable preservation agents are for example parabens and phenoxyethanol. These preservation agents can be used alone or in combination with each other. The compositions intended for hair or scalp care may contain arctic bramble cell culture, preferably 0.001 to 10 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 2 % by weight. Further, the compositions intended for hair or scalp care may also contain different caring agents. These caring agents are typically in the amount of 0.1 to 40 % by weight. Preferably the caring agent content of a product is 0.1 to 20 % by weight. The compositions intended for hair or scalp care may contain in addition to arctic bramble cell culture, also other substances acceptable in the field of cosmetics, like cationically active substances, such as cetrimonium chloride, in addition an emulsion forming substance, such as for example cetyl alcohol, cetearyl alcohol, ceteareth-20. In addition, the compositions intended for hair or scalp care can contain one or more adjuvants acceptable in the field of cosmetics, such as a cellulose derivative, ethanol and/or water. Also, oils, waxes and fatty alcohols can be present in the compositions, according to the invention, intended for hair or scalp care.

The serum compositions may contain arctic bramble cell culture preferably 0.001 to 25 % by weight, more preferably 0.01 to 5 % by weight and most preferably 0.01 to 2 % by weight.

The serum compositions may contain also other substances acceptable in the field of cosmetics such as emulsifying agents, chelating agents, solvents, preservatives, stabilizers together with substances affecting the skin permeability of the composition. These substances may be e.g. methylpropanediol, glycerin, phenoxyethanol, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, xanthan gum, propanediol, ammonium acryloyldimethyltaurate/VP copolymer, polyisobutene, disodium EDTA, lecithin, glucose, hydrogenated phosphatidylcholine, inulin lauryl carbamate, PEG-7, trimethylolpropane coconut ether, chondrus crispus, ethyl pyrrolidone, and cellulose gum. Additionally, the serum compositions may contain different moisturizing and skin conditioning agents such as heptyl undecylenate, ethylhexylglycerin, caprylyl glycol, ethylhexyl cocoate, peat extract and glycolipids. As indicated, the arctic bramble cell culture is preferably used in the form of freeze-dried powder, and the amounts given above are calculated for said powder. However, in case methanol or ethanol extracts are used, it is within the expertise of a person skilled in the art to adapt the amounts correspondingly.

The phenolic profile of the arctic bramble cell cultures of the present invention is unique and different compared to the arctic bramble berry fruit, as said cell cultures may contain procyanidins, gallic acid derivatives, kampherol derivatives, quercetin derivatives, vitamin

E (a-tocopherol), unique carbohydrate composition and unique fatty acid composition.

Some of the culture lines produce high amounts of natural colorants, i.e. anthocyanins.

The cell cultures also contain natural flavors and lipids with beneficial fatty acid composition, which are desired ingredients in cosmetic and hygiene preparations. The cell cultures can be generated around the year at different scales in consistent quality, which can be constantly monitored. It was surprisingly found that several advantageous effects may be achieved with the present invention.

Examples

The following examples are illustrative embodiments of the present invention, as described above, and they are not meant to limit the invention in any way.

Example 1

GENERATING ARCTIC BRAMBLE SUSPENSION CELL CULTURE

0.5-1 g of callus line obtained from arctic bramble plant material (in vitro grown arctic bramble plant), with red-color was transferred in 10-20 ml of liquid MS medium, pH 4.0 with 0.46 μΜ kinetin, 5.37 μΜ NAA (a-naphthaleneacetic acid) for initiation of suspension cultures. The suspended callus cells were cultivated at 23 - 25°C under day-night illumination regime (photoperiod 16: 8 h, irradiation 40 μιηοΙ m^~2s ^_1) in Erienmayer shake flasks using a shaker at 90 - 120 rpm. The cultures were sub-cultured after 11 +/- 3 days by diluting cultures 1 : 3 with the fresh medium, and the clumps of the callus were removed. After 4 - 8 sub-culturing the cell lines in suspension were used as inoculum in bioreactor up-scaling.

Example 2

CULTIVATION OF ARCTIC BRAMBLE CELL CULTURE IN BIOREACTOR WITH/WITHOUT LED -ILLUMINATION

The inoculum was red-coloured arctic bramble suspension cell culture (KAS 341/15), obtained from arctic bramble plant material with a method consisting of formation of calli and generation of suspension cell culture by favouring dark red coloured calli, as described in example 1. Said arctic bramble suspension cell culture was used as inoculum in glass bioreactors under illumination of LED light panel with light intensity of 860 Ix, colour temperature 2700-3000K, spectral range of 450-780 nm, with at least 25 % of the emission in the range of 630-760 nm (fermenter A). A reference study was conducted with normal light of 120 Ix in pilot hall (fermenter B).

Inoculum in both fermenters A and B comprising of arctic bramble cells (KAS 341/15) was made up to with the fresh MS medium described in example 1, to obtain initial cell density of 30 g L^"1, for bio-reactors A and B having the volume of 2 L. The suspensions were grown in the both bio-reactors for 9 days. The cultivation conditions were the following : temperature 25°C, dissolved oxygen (DO) over 20 %, agitation speed 100-300 rpm, pH was followed but not controlled, aeration with pressurized air 1.5 l/min, continuous expose to light (LED or normal). Sampling was carried out after 3 and 6 days, where the viability of the cells was tested with FDA (Fluorescein Diacetate) staining. After 9 days period the viability of the final culture was measured by FDA, and the cells were separated from the liquid medium on Miracloth filtration paper by vacuum Buchner filtration. The fresh weight (FW) of the cells was measured, and after freeze-drying of the cells the dry weight (DW) was obtained. In table 1 viability, cell color, FW (fresh weight) and DW (dry weight) are presented.

Table 1.

A - with LED light panel

B - with pilot hall background light

Both cultures grew very well and the viability stayed at over 95%. However, the LED illumination increased significantly the intensity of red colour in the bioreactor during 9 d cultivation. The dry weight of the biomass yield was higher in the LED illuminated fermenter, which means that the cells cultivated in LED light were more intact, whereas the cells with less light had higher water content in the cells. Anthocyanins and phenolic compounds were analysed in the dry biomass by UHPLC-DAD- MS/MS of both tests A and B. The results are presented in following table 2.

Table 2.

* procyanidins have polymerization degree of 13.

Figure 1 shows anthocyanin contents (peak at about 3.1) of arctic bramble cell cultures obtained with LED irradiation (A) and without it (B).

Claims

1. A method for producing artic bramble cell culture, characterized in that the method comprises two stages, where in the first stage arctic bramble callus line obtained from callus produced from sterile pieces of in vitro grown arctic bramble plants is cultured in a liquid medium comprising a medium used for plant cell and tissue culture, under day-night irradiation regime under normal light or under irradiation using LED light carried out as irradiation using LED light carried out as intermitting irradiation or as constant irradiation, to obtain primary arctic bramble suspension cell culture, and in the second stage the primary arctic bramble suspension cell culture is up-scaled in at least two steps in the liquid medium; and irradiation with LED light is used in the first stage or in the second stage or in both stages; and where the second stage comprises more than two steps, the second step may be carried out exposed to no irradiation.

2. The method according to claim 1, characterized in that in the second stage the primary arctic bramble suspension cell culture is up-scaled in at least three steps.

3. The method according to claim 1 or 2, characterized in in the second stage the primary arctic bramble suspension cell culture is up-scaled in at least four steps.

4. The method according to any one of claims 1- 3 characterized in that the liquid medium is modified MS medium comprising at least one component selected from sucrose, agar, gelrite, plant growth regulators and combinations thereof, and the pH of modified MS medium is adjusted into the range of 3-7.

5. The method according to claim 4, characterized in that plant growth regulator is selected from cytokines, auxins and combinations thereof.

6. The method according to any one of claims 1- 5, characterized in that the LED light has color temperature of 2700-3000K.

7. The method according to any one of claims 1- 6, characterized in that the LED light comprises an effectual spectrum comprising at least 25 % of an integral in the wave length range of 630 nm-760 nm, calculated from the total effectual spectrum.

8. The method according to any one of claims 1- 7, characterized in that the irradiation source is LED light with illumination intensity over 500 Ix, preferable over 800 Ix.

9. Arctic bramble cell culture obtained by the method according to any one of claims 1-8, wherein said cell culture comprises 0.1-10 wt% (dry weight) of anthocyanins and 0.1-5 wt% (dry weight) of proanthocyanidins.

10. Composition comprising the arctic bramble cell culture according to claim 9, characterized in that the composition is selected from cosmetic compositions, hygiene and personal care products, foods, feeds, pet foods, nutritional compositions, nutraceutical compositions, and pharmaceutical compositions.

11. The composition according to claim 10, characterized in that the cosmetic composition is selected from day creams, night creams, foundation creams, peeling creams, lipsticks, colour cosmetics, skin serums, mascaras, products for hair and/or scalp care, washing products for skin or hair, and skin hygiene products.

12. The composition according to claim 11, characterized in that the cosmetic composition comprises the arctic bramble cell culture in powder form in an amount of 0.001 to 25 % by weight, more preferably the amount of the powder is 0.01 to 5 % by weight, and most preferably 0.01-1 % by weight.

13. Use of the arctic bramble cell culture according to claim 9 in cosmetic compositions, hygiene and personal care products, in foods, feeds, pet foods, nutritional compositions, nutraceutical compositions, and pharmaceutical compositions, and for providing aroma, flavor and/or color to the products.